The intracellular pathogen Francisella tularensis (FT) is one of the most virulent bacterial pathogens known, and is capable of infecting humans and many animals. FT could potentially be a dangerous bioweapon; understanding its pathogenesis and developing an effective vaccine for tularemia have therefore become increasingly urgent goals. Nonetheless, remarkably little is known about the pathogenesis of tularemia. One strategy that bacterial pathogens use to enhance their survival during infection is phase or antigenic variation of surface components, which can allow evasion of host defenses. FT shows antigenic variation of lipopolysaccharide (LPS), switching between colony variants with different LPS species: one LPS fails to stimulate nitric oxide (NO) production by rat macrophages, so that the intracellular bacteria survive and grow; LPS of the other variant triggers NO production and efficient killing of the bacteria. The two LPS structures have not been characterized, and nothing is known of the regulation of LPS variation or its role in FT pathogenesis. The long-term goal of this project is to determine how antigenic variation of LPS, and possibly of other components, contributes to the pathogenesis of tularemia and to the ability of FT to evade innate and acquired host defenses. Aim 1 is to initiate studies on LPS antigenic variation by characterizing the structures of the LPS species, identifying the relevant genetic loci, determining the molecular mechanism of antigenic variation, and assessing the effect of LPS variation on the interaction of FT with human cells. It is likely that LPS is not the only antigenically variable component of FT. The FT genome contains a number of genes with the repetitive DNA sequences that are characteristic of phase variable genes in other organisms. Aim 2 is to determine if expression of those genes is indeed subject to phase variation. These studies will provide an initial assessment of the potentially important contribution that antigenic variation makes to the pathogenesis of tularemia.